Macrophages (Mphi) loaded with free cholesterol (PC) and phospholipid (PL) are prominent in atherosclerotic lesions, and events associated with the necrosis of these cells appear to be a least partly responsible for lesion progression. The major goal of this proposal is to explore several hypotheses that relate lipid changes in Mphi wit foam cell necrosis and lesion progression. Preliminary studies have demonstrated that FC loading of cultured Mphi initially leads to an increase in cellular phosphatidylcholine (PC) due to stimulation of CTP:phosphocholine cytidylyltransferase (CT), which probably represents an adaptive response to av awed the toxicity of high PC:PL ratios. After long-term PC loading, however, the PL content of the cultured Mphi begins to decrease, and the cells subsequently show signs of toxicity. In this context, Aim #1 will explore several specific ideas regarding the molecular mechanisms responsible for the initial up-regulation of CT in FC-loaded Mphi. In Aim #2, the biochemical and morphological properties associated with the toxicity of long-term FC-loaded Mphi in culture will be defined, and manipulations of PL metabolism in these cells will be used to explore several specific hypotheses relating Pl changes in these cells to cytotoxicity. These hypotheses are; (a) a toxic cellular FC:PL ratio, resulting from an eventual decrease in cellular pl mass, leads to Mphi toxicity and necrosis; (b) the generation of lysoPC, resulting from the exposure of the excess Mphi PL to phospholipase, leads to Mphi toxicity and necrosis; and (c) the generation of ceramide, resulting from the exposure of the excess Mphi sphingomyelin to sphingomyelinases, leads to Mphi apoptosis. The goal of Aim #3 is to explore these cytotoxicity hypotheses in a physiological setting using mouse models of atherosclerosis. First, lesion of atherosclerosis-susceptible apo E knockout (EO) mice will be carefully examined to determine if increases in FC:PL ratio, lysoPC, and/or ceramide precede the onset of lesion Mphi necrosis. Then, CT transgenic mice and Mphi-specific or heterozygous CT knockout mice will be created and crossed with EO mice to show that manipulations of PC metabolism can affect lesion necrosis and progression in vivo.